Abstract Current treatment of head and neck squamous cell carcinoma (HNSCC) with immune checkpoint inhibitors and radiotherapy (RT) are initially effective, but relapse is common. The goal of our research is to understand mechanisms of response and resistance and to use this knowledge to improve therapy. We show that during the response phase to treatment, RT increases STAT1 phosphorylation, CXCL9/10 secretion, and PDL1 expression on cancer cells, and increases CD8 and CD4 infiltration and activation. These effects, however, dissipate in the resistance phase, and a significant increase in regulatory T cells (Tregs), STAT3, and TGF?1 are observed. Treg depletion and anti-STAT3 targeting restores response to RT and anti-PDL1, resulting in suppression of plasma levels of TGF?1, activation of T effector cells (Teff, Cd44+, IFN-G+ CD4 and CD8 T cells) and complete tumor eradication in an orthotopic model of HNSCC. This effect is unachievable with the addition of anti-CTLA4, anti-Tim3, or with hypofractionation of RT. Our in vitro preliminary data show that RT enhances STAT3 phosphorylation on CD4 T cells, leading to increased Treg conversion. This is reversed with STAT3 inhibitors. CD4 T cell conversion toward a Treg phenotype represents an important mechanism of immune evasion. TGF?1 has been established as a necessary growth factor for conversion of nave CD4 T cells to Tregs through induction of FoxP3 expression. In addition, STAT3 is a necessary transcription co-factor for FoxP3 expression. Our central hypothesis is that during the response phase, RT induces a distinct cancer cell STAT1-mediated CXCL9/10 secretion which recruits and activates Teff resulting in enhanced synergy with anti-PDL1. RT, however, also promotes CD4 conversion to Treg cells by phosphorylation of STAT3, which leads to the development of treatment resistance by inhibiting Teff function. Using knockdown constructs of STAT1 and CXCL9/10 on cancer cells and a CXCR3-/- mouse model, we will determine the effect of RT-induced chemokines in Teff chemotaxis, proliferation, and TCR activation (Aim1). To determine how RT affects CD4 T cell conversion and how that feeds back to negatively affect Teff function, we will use the CD4-Cre/ Stat3flox/flox mice with knockdown of STAT3 on CD4 T cells as well as STAT3 inhibitors (Aim2). Finally, we will use tissue and blood samples from a Phase I clinical trial aimed at using neoadjuvant combination RT and anti-PDL1 and will examine whether that changes the immune landscape to increase Teff/Treg ratio and enhance Teff activation profile (Aim3). We expect these studies to elucidate molecular and cellular parameters of RT plus immune checkpoint blockade and to help develop more effective therapy for HNSCC.